Effect of Ultraviolet-B Radiating Drosophila melanogaster as Host on the Quality of Trichopria drosophilae, a Pupal Parasitoid of Drosophila suzukii

Simple Summary The mass rearing of parasitoids is a basic prerequisite for their use in biological control. If parasitoid rearing is effective, host/parasitoid mixtures will not form. In this study, we used an irradiated Drosophila melanogaster host to supply parasitoids and assessed whether all adults that emerged after parasitization were Trichopria drosophilae. The results of this study showed that irradiation of host pupae for 6 h is the preferred treatment for promoting the emergence and therefore the potential application of T. drosophilae in the control of Drosophila pests. At the same time, the results also serve as an important reference for reducing costs and improving the efficiency of biological pest control. Abstract The pupal parasitoid, Trichopria drosophilae Perkins (Hymenoptera: Diapriidae), is an ectoparasitoid of the genus Drosophila with great potential for application in biological control based on its excellent control efficiency for Drosophila suzukii Matsumura (Diptera: Drosophilidae), and it has has even been commercialized by biofactories. Due to its characteristics of short life cycle, large number of offspring, easy rearing, rapid reproduction, and low cost, Drosophila melanogaster (Diptera: Drosophilidae) is currently being utilized as a host to mass produce T. drosophilae. To simplify the mass rearing process and omit the separation of hosts and parasitoids, ultraviolet-B (UVB) was used as an irradiation source to irradiate D. melanogaster pupae, and the effects on T. drosophilae were studied. The results showed that UVB radiation significantly reduces host emergence and affects the duration of parasitoid development (female: F0 increased from 21.50 to 25.80, F1 from 23.10 to 26.10; male: F0 decreased from 17.00 to 14.10, F1 from 17.20 to 14.70), which has great significance for the separation of hosts and parasitoids as well as of females and males. Of the various studied conditions, UVB irradiation was ideal when the host was supplied with parasitoids for 6 h. The selection test results showed that the female-to-male ratio of emerging parasitoids in this treatment was highest at 3.47. The no-selection test resulted in the highest rates of parasitization and parasitoid emergence rate, maximized inhibition of host development, and allowed the omission of the separation step. Finally, the results of the semi-field test showed that the parasitoids bred in this treatment could search for their hosts normally and could therefore be directly applied in the biological control of Drosophila pests in the field.


Introduction
Drosophila melanogaster Meigen (Diptera: Drosophilidae) is well known as one of the most intensively studied model insects [1,2]. In contrast to this saprophageous species, degree of teratogenicity in offspring while affecting their phenotype and behaviour [35][36][37][38]. At the same time, ultraviolet radiation enhances the parasitic effect of parasitoids on their hosts in many ways. Ultraviolet radiation affects the selection and suitability of hosts for parasitoids, which in turn impacts the effectiveness of parasitoids [39][40][41][42][43][44][45]. The parasitization of Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) was shown to be significantly increased in ultraviolet-irradiated hosts compared to unirradiated host eggs. There was also a significant increase in T. chilonis emergence from hosts, and radiation inhibited the development of unparasitized hosts. In addition, compared with gamma irradiation, the host treated with ultraviolet radiation is more likely to be parasitized, and ultraviolet radiation treatment is more suitable for rearing and extension applications of biological natural enemies [42,46]. If ultraviolet irradiation can achieve lower levels of host emergence, in addition to its features of host suitability and parasitoid efficiency, this would result in greatly improved rearing efficiency.
This study aims to determine the optimal host irradiation dose to achieve the highest parasitism rate in simulated outdoor field environment conditions. The offspring parasitoids are obtained using irradiated pupae as hosts, and the parasitized environment under natural conditions is restored to the maximum extent, so as to evaluate their effectiveness. After a series of experiments on the effects of ultraviolet radiation on hosts, UVB was selected as a suitable test radiation for irradiating hosts [35]. In this study, D. melanogaster pupae irradiated with different doses of UVB were used as hosts to rear T. drosophilae with assessment of the parasitism rate, emergence rate, sex ratio, pupal death rate, development duration, longevity, and outdoor parasitization performance of T. drosophilae. This research will provide compelling support for the mass rearing of natural enemy resources and simplify the rearing process. It will also provide basic information on the effects of the use of ultraviolet-irradiated hosts on the development of parasitoids.

Insects
Laboratory colonies of D. melanogaster and T. drosophilae were collected from ecological orchards (119 • 13 39.98 E, 25 • 42 36.00 N) in Fuqing City, Fuzhou City, Fujian Province, P.R. China. The trap box was a square plastic box with an escape-prevention device and the exterior of the box was wrapped with alternating black, red, and yellow tape to attract flies [47][48][49][50]. The box contained fruit that had just started to rot and an artificial diet for flies. At the same time, T. drosophilae were trapped using a box containing a skimmed cotton ball soaked with honey and pupae of D. suzukii and D. melanogaster.
The collected insects were reared indoors and maintained under controlled conditions (25 ± 1 • C, 65 ± 5% RH, 12:12 h (L/D)). In order to ensure fresh food and clean water and to avoid mildew adversely affecting insect sources, the food and water were kept adequately well supplied and replaced regularly. The gauze cage was covered on all six sides with 100-mesh nylon that had a cylindrical operating cuff (L: 30.00 cm, D: 10.00 cm) on one side to facilitate handling inside the cage. Adult D. melanogaster were maintained in cages and supplemented with a standard cornmeal-based artificial diet. The artificial diet formula contained cornmeal: 50.00 g, sucrose: 40.00 g, brewery yeast: 20.00 g, agar: 5.00 g, 36% v/v acetic acid: 3.00 mL, 95% v/v ethanol: 7.00 mL, ethyl p-hydroxybenzoate: 1.00 g, and sodium benzoate: 1.00 g [17][18][19]. The pupae of D. melanogaster were placed in a T. drosophilae cage for 24 h and then placed in a new cage until they emerged. The nutriment of T. drosophilae adult was fresh 10% honey water.

UVB Radiation
UVB lights (Nanjing Huaqiang Electronics Co., Ltd., Nanjing, China), which could emit 300 nm UVB, were used as the source to irradiate newly collected pupae for different durations at 50.00 cm above the pupae.

Host Species Preference
A selection test was designed to evaluate whether T. drosophilae preferred to parasitize treated D. melanogaster pupae. Firstly, newly formed D. melanogaster pupae (within 24 h) were taken, and the collected pupae were placed in Petri dishes lined with moistened filter paper and irradiated to UVB for 0, 3, 6, and 9 h. From each test group corresponding to 4 treatments, 25 pupae were taken, with 100 pupae taken in total. The pupae were placed on a designated quarter of a moistened filter paper in a Petri dish (d: 9.00 cm, h: 2.00 cm) using the cross-division method and provided with 10 pairs (female/male = 1:1) of sexually mature virgin T. drosophilae for 24 h. Each treatment included five replicates. After parasitization, the pupae of the four treatments were separated. The number and sex of parasitoids that emerged in each treatment were counted separately and used to calculate the number of emerged parasitoids and the female-to-male sex ratio.

Parasitic Efficiency Based on Generations
A no-selection test aimed to evaluate the parasitic ability of T. drosophilae on differently treated fly pupae. First, newly formed D. melanogaster pupae (within 24 h) were taken, and the collected pupae were placed in Petri dishes lined with moistened filter paper and irradiated to UVB for 3, 5, 6, 7, and 9 h. An additional negative control was set up without radiation. Irradiated flies pupae were placed individually in Petri dishes lined with moist filter paper and provided with 10 pairs (female/male = 1:1) of sexually mature virgin T. drosophilae for 24 h. One hundred pupae were taken from each treatment and each treatment included three replicates. This generation treatment process was recorded as F0.
To wait for the emergence, 100 treated pupae were placed in a Petri dish. The number and sex of parasitoids were recorded daily and used to calculate the emergence rate. Pupae from which an adult (either parasitoid of fly) did not emerge were dissected to identify whether the pupae contained parasitoids or flies. The sex of parasitoids was considered based on the type of antennae or the presence of an ovipositor on the abdomen, and the amount corresponding to each sex was counted. The above data were used to calculate the parasitism rate and sex ratio (female/male). Pupae that did not emerge and died of blackening were recorded as the number of dead pupae and were used to calculate the pupal death rate. The development duration of parasitoids was from the completion of treatment to the emergence of parasitoids. At the same time, newly emerging T. drosophilae were randomly selected from each treatment and provided with honey and water but no host under controlled conditions (25 ± 1 • C, 65 ± 5% RH, 12:12 h (L/D)). The survival conditions of T. drosophilae were recorded every day to calculate their lifespan and the dead T. drosophilae were cleaned up at the same time. Ten adults of each sex were used in each replicate of the development duration and longevity test. A certain number of newly emerged male and female parasitoids were randomly selected from each treatment, and the above-described no-selection experiments were repeated, with the same statistical indicators and computational methods as above. This generation treatment process is recorded as F1.

Outdoor Test
The newly formed pupae of D. melanogaster (within 24 h) were treated with UVB for 6 h and then served as hosts. The proportion of pupae to female parasitoids was 1:10, and sexually mature virgin T. drosophilae were provided for 12 h of outdoor parasitization. A negative control without radiation was also set up, and each treatment included three replicates. After parasitization, the pupae were placed in the parasitoid rearing room for the emergence of parasitoids, recorded as F1. The parasitoids were separately packed and supplemented with honey water and kept until the parasitoids were sexually mature. After normal pupae were provided and parasitized in accordance with the above proportion, the emerging parasitoids were recorded as F2.
The outdoor test was carried out in a bioassay cage (30.00 cm × 30.00 cm × 30.00 cm, covered on all six sides with 100 mesh nylon) with the following conditions: a surface lined with 100 pupae of halved cut grapes for parasitoids and cotton moistened with honey water as a nutritional supplement for the parasitoids. Twenty sexually mature virgin parasitoids (female/male = 1:1) were released into cages, grapes were collected after 12 h, and the pupae were brushed onto Petri dishes lined with moistened filter paper, and emergence was awaited. Both generations were tested. The measures of F1 and F2 parasitoids quality for outdoor testing and calculations were the same as the no-selection test in Section 2.4.

Data Analysis
WPS Office 2022 (Kingsoft Co., Ltd., Beijing, China) was used to count raw data and calculate parasitism rate, emergence rate, sex ratio, pupal death rate, development duration, and longevity, and also for plotting. All data were analyzed using one-way ANOVA with SPSS v.23.0 (SPSS Inc., Chicago, IL, USA), and multiple comparisons were performed using the LSD method. Statistical results were expressed as mean ± SD, with p < 0.05 considered to indicate statistical significance, and multiple comparison results were marked according to the letter-marking method.

Host Species Preference
In the selection test, there was no significant difference in the number of females (F 3, 16 = 1.216, p = 0.336) and males (F 3, 16 = 1.280, p = 0.315) that emerged between all treated hosts. The test results show that parasitoids preferred the host pupae of B3 (UVB and irradiation hours; similar below), from which 16.80 parasitoids emerged, corresponding to an emergence rate of 67.20%. The numbers of females and males that emerged were 11.20 and 5.40, respectively, which was not significantly different from the control (p = 0.179, p = 0.766). Meanwhile, 14.20 parasitoids emerged from T. drosophilae parasitized hosts of B9, an emergence rate of 56.80%. Of these, there were 10.00 females and 4.20 males, both of which were not significantly different from the control (p = 0.492, p = 0.554, Figure 1). emergence of parasitoids, recorded as F1. The parasitoids were separately packed and supplemented with honey water and kept until the parasitoids were sexually mature. After normal pupae were provided and parasitized in accordance with the above proportion, the emerging parasitoids were recorded as F2.
The outdoor test was carried out in a bioassay cage (30.00 cm × 30.00 cm × 30.00 cm, covered on all six sides with 100 mesh nylon) with the following conditions: a surface lined with 100 pupae of halved cut grapes for parasitoids and cotton moistened with honey water as a nutritional supplement for the parasitoids. Twenty sexually mature virgin parasitoids (female/male = 1 : 1) were released into cages, grapes were collected after 12 h, and the pupae were brushed onto Petri dishes lined with moistened filter paper, and emergence was awaited. Both generations were tested. The measures of F1 and F2 parasitoids quality for outdoor testing and calculations were the same as the no-selection test in Section 2.4.

Data Analysis
WPS Office 2022 (Kingsoft Co., Ltd., Beijing, China) was used to count raw data and calculate parasitism rate, emergence rate, sex ratio, pupal death rate, development duration, and longevity, and also for plotting. All data were analyzed using one-way ANOVA with SPSS v.23.0 (SPSS Inc., Chicago, IL, USA), and multiple comparisons were performed using the LSD method. Statistical results were expressed as mean ± SD, with p < 0.05 considered to indicate statistical significance, and multiple comparison results were marked according to the letter-marking method.

Host Species Preference
In the selection test, there was no significant difference in the number of females (F3, 16 = 1.216, p = 0.336) and males (F3, 16 = 1.280, p = 0.315) that emerged between all treated hosts. The test results show that parasitoids preferred the host pupae of B3 (UVB and irradiation hours; similar below), from which 16.80 parasitoids emerged, corresponding to an emergence rate of 67.20%. The numbers of females and males that emerged were 11.20 and 5.40, respectively, which was not significantly different from the control (p = 0.179, p = 0.766). Meanwhile, 14.20 parasitoids emerged from T. drosophilae parasitized hosts of B9, an emergence rate of 56.80%. Of these, there were 10.00 females and 4.20 males, both of which were not significantly different from the control (p = 0.492, p = 0.554, Figure  1). Here and below, bars refer to mean ± SE and different lowercase letters above the bars indicate significant differences (Duncan's LSD, p < 0.05). CK means control with no irradiated treatment.
In the selective host test, there was no significant difference in sex ratios of the parasitoids that emerged from differently treated hosts (F 3, 16 = 1.396, p = 0.280). The sex ratios of parasitoids that emerged from B6, B9, and B3 were 3.47, 3.25, and 2.40, respectively, all of which were higher than but not significantly different from the control (p = 0.150, p = 0.554, p = 0.766, Figure 2). significant differences (Duncan's LSD, p < 0.05). CK means control with no irradiated treatment.
In the selective host test, there was no significant difference in sex ratios of the parasitoids that emerged from differently treated hosts (F3, 16 = 1.396, p = 0.280). The sex ratios of parasitoids that emerged from B6, B9, and B3 were 3.47, 3.25, and 2.40, respectively, all of which were higher than but not significantly different from the control (p = 0.150, p = 0.554, p = 0.766, Figure 2).

Discussion
A number of studies on the effects of radiation on organisms have been applied to insects because of their easy handling, short life cycle, large number of offspring produced by most species, and other favorable biological characteristics [51]. These processes include traditional gamma rays, emerging ultraviolet rays, and microwaves [52][53][54][55]. Ultraviolet radiation affects the adaptation of eggs, larvae, pupae, and adult stages of insects, encompassing all periods of insect development and showing great potential for application in integrated pest management [42,56]. The ultraviolet part of the spectrum is widely used for sterilization, surface disinfection of insect eggs, insect attraction and extermination, and inhibition of different stages of the insect life cycle [57][58][59][60]. Insects may be at high risk from ultraviolet radiation due to their small size. Radiation may penetrate much more deeply in insect tissues than larger organisms, thus disproportionately affecting their performance [36].
Trichopria drosophilae is widely used in the biological control of flies, and it shows great flexibility in host selection [26,28,61,62]. Host species, age, and treatment methods can significantly affect parasitization of the parasitoids [63,64]. Our study is the first to both assess the suitability of T. drosophilae for UVB radiation of hosts and carry out quality test evaluation via an outdoor test. The results show that UVB radiation can affect the parasitic efficiency and performance of T. drosophilae, which can influence the suitability of hosts for parasitoids. Similar studies have shown that Trichogramma chilonis prefers to parasitize ultraviolet-irradiated host eggs compared to fertilized and unfertilized eggs. Therefore, irradiation improves the adaptability of parasitoids to hosts [43]. Furthermore, the emergence rate of 1-day-old pupae of Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) was significantly decreased by UVB irradiation [65]. This effect may be caused by the changes in the host immune system under the influence of ultraviolet radiation during the co-evolution of the hosts and parasitoids [66]. Ultraviolet radiation can kill hosts in a certain stage and retain host nutrients for the growth and development of parasitoids, which may partially explain why parasitoids prefer to parasitize hosts after ultraviolet radiation [43]. Our selection test showed that more parasitoids emerged from hosts treated with short UVB, but the difference was not significant with others. Combining the result of the ratio of females to males in the selection test with previous studies on irradiated hosts, we can infer that B6 should be a more suitable means of treating hosts [35]. The host treated with UVB was parasitized by T. drosophilae, and the parasitism rate was higher than the emergence rate. A small proportion of parasitized hosts did not successfully emerge from T. drosophilae, probably because the short radiation of hosts had an effect on the growth and development of parasitoids within the host. Some T. drosophilae failed to become normal individuals, or their development was impaired, or they were even deformed, thus preventing some of them from successfully emerging. If the test host is D. suzukii, with a strong immune response, it is worth investigating whether this loss can be reduced to a certain extent in future experiments. Doing so will pave the way for the successful application of this technique to more species of Drosophila pest control. The use of microscopy in this study revealed that a significantly lower number of T. drosophilae in the treatment group were incompletely developed, but this was even lower in the control. The increased rate of dead pupae after ultraviolet radiation also indicates that the growth and development of the host and T. drosophilae are affected by treatment to a certain extent. Meanwhile, radiation increased the parasitic efficiency of parasitoids on their hosts. Unfortunately, after two generations of testing, this increase in efficiency was not passed to the next generation. A scientific question worth investigating is whether successive generations of irradiated treated hosts supplying parasitoids with parasitization will allow this tendency to be passed to future generations. With the increase in irradiation time from 3 h to 5 h in the no-selection test, the mortality of host pupae gradually increased. However, this trend almost completely disappeared later as the treatment time continued to increase.
A follow-up test should be conducted to explore whether the pupae have a certain tolerance or upper limit to irradiation. The longevity of male and female parasitoids of B5 was lower than B3 and B6. Whether this treatment duration could be used as a special method to affect the survival of parasitoids needs to be further tested to verify its feasibility.
The connections between insects and microbes are wide-ranging and influential [67]. Among the adaptive effects of microbes on hosts, defense against natural enemies is increasingly considered to be universal, especially in those associations involving heritable and facultative bacteria [68]. The study of the interaction between Drosophila, bacteria (Spiroplasma MSRO), and parasitoids has laid the foundation for our understanding of the important roles played by each part of symbionts. Moreover, MSRO is also a male killer. In this study, the developmental trends of female and male parasitoids were in stark contrast. Whether treatment affected the survival of microbes and their interactions and then affected the different adaptability of parasitoids of different sexes to treatment is a question to be scientifically explored [69]. Maternally inherited bacteria, meanwhile, are common in many insects but are generally unculturable, maintaining their interests by manipulating their host to reproduce or giving it an adaptive advantage [68]. This benefit may depend on the environment to resist abiotic stress or natural enemies. It remains to be explored whether the radiation treatment in this study will affect their survival and further prolong the development duration of female parasitoids. After UVB radiation, males and females showed different developmental trends, indicating that they had different adaptations to UVB irradiation. The adaptation of males to radiation compared with females is important in shortening the duration of the development of T. drosophilae and guiding the separation of males and females during mass rearing.
As an important indicator of parasitoids for biological control, obtaining as many females as possible can improve the efficiency of pest control, and it is also an important guide for the mass rearing of parasitoids [70][71][72][73]. The sex ratio of UVB-irradiated hosts supplied with T. drosophilae increased with radiation time to a maximum of 7 h. Changing temperatures are also a key factor for the effectiveness of control when carrying out field applications of parasitoids. At the same time, temperature is also an important factor in the sex ratio [74]. A temperature of 26 • C is the highest reproductive temperature for the offspring sex ratio of Spalangia endius Walker (Hymenoptera: Pteromalidae) [75]. The sex ratio of Diversinervus elegans Silvestri (Hymenoptera: Encyrtidae) decreased gradually with the increase in temperature in the range of 18-30 • C, and the female ratio of its progeny reached 74.24% under 18 • C [76]. In our study, UVB radiation was observed to affect the sex ratio of parasitoids and is thus a potential method for female acquisition. Taken together, the increase in the sex ratio due to radiation seems to have been at the expense of male death or non-emergence [77]. This combination of low temperature and UVB radiation leading to parasitoid feminization is significant for guiding irradiation of pupae in mass rearing parasitoids for field applications, and it is worth further investigating the treatments selected in this study in combination with low-temperature refrigeration techniques [40,41,51,78]. The mechanisms controlling the sex ratio of parasitoids are extremely complex, and they include adult parasitoid age, mating status, nutrient supply, genetics, host, and environment, which all affect the sex ratio of parasitoids [79][80][81][82]. This study only investigated the effects of ultraviolet radiation and outdoor environmental conditions on the sex ratio of parasitoids, while the interaction between other factors and underlying mechanisms needs to be further explored [83][84][85][86][87][88]. This study reveals their comprehensive influence on the sex ratio and its internal mechanism.
The results of the outdoor quality tests of F1 and F2 generations of T. drosophilae show that approximately the same quality of parasitoids emerged from irradiated and normal pupae. The index values were higher for most treatments than for the control, indicating that irradiated hosts are able to successfully search for hosts and complete normal parasitization before emerging under outdoor test conditions and can thus be directly applied in field prevention and control. However, there are also a lot of criticisms, and it will be very important to address these in future work. As a rather important aspect, irradiation also has certain drawbacks; for example, the pupae of flies have a high death rate after radiation. In the process of mass rearing parasitoids, a high rate of dead pupae will increase reproduction cost [89][90][91]. At the same time, the collected pupae will be wasted, and reproduction efficiency will be reduced [92]. If we can streamline the collection of pupae from D. suzukii, we can then consider using it as a host to try to minimize these losses. At the same time, the host range available for large-scale rearing of parasitoids will be expanded, which maximizes the effectiveness of biological control and minimizes the cost. Therefore, all aspects need to be taken into account when evaluating and selecting radiation conditions. It is expected that the process can be streamlined while improving reproductive efficiency and economic benefits. We expect to realize the vision of providing more highquality and efficient parasitoids with great outdoor adaptability for the biological control of Drosophila.

Conclusions
Based on the results of this study, and as described above, ultraviolet radiation was shown to affect the parasitic efficacy and outdoor performance of T. drosophilae. The research results provide a theoretical basis for further clarification of the application of ultraviolet irradiation in the field of pest control. Testing the outdoor performance of parasitoids can unlock the full potential of resources for the biological control of pest. It also can optimize long-lasting pest control strategies in the field to protect the environment and achieve sustainable development.
Author Contributions: Methodology, X.L. and Q.J.; performed the experiments, X.L., Y.Y., Q.F. and Q.Z.; writing-original draft preparation, X.L.; writing-review and editing, X.L. and Q.J.; supervision, Q.J. All authors have read and agreed to the published version of the manuscript.